KR20080098636A - Pharmaceutical latrunculin formulations - Google Patents

Abstract

The present invention relates to an aqueous pharmaceutical formulation comprising at least one latrunculin and the formulation does not contain a substantial amount of dimethyl sulfoxide. In one embodiment, the present invention is directed to an aqueous pharmaceutical formulation comprising at least one latrunculin in an amount of 0.001-2% w/v, a non-ionic surfactant in an amount of 0.01-2% w/v, and a tonicity agent to maintain a tonicity between 200-400 mOsm/kG, at a pH between 4 to 8, wherein the latrunculin, the surfactant, and the tonicity agent are compatible in the formulation, and the formulation does not contain a substantial amount of dimethyl sulfoxide. The formulation is stable for at least six month at refrigerated temperature. The present invention further provides a method of reducing intraocular pressure, a method of treating glaucoma, a method of inhibiting wound healing after trabeculectomy, and a method of inhibiting angiogenesis.

Description

Pharmaceutical Lathrunculin Formulations {PHARMACEUTICAL LATRUNCULIN FORMULATIONS}

The present invention relates to pharmaceutical formulations, in particular ophthalmic formulations of macrocyclic cytoskeletal active compounds, such as ratunculin compounds and their related analogs. The invention also relates to the prevention or treatment of diseases or disorders affected by variations in the maintenance of actin cytoskeleton, for example in the treatment of disorders of elevated intraocular pressure, such as primary open angle glaucoma and optic nerve protection in humans and other mammals. It relates to a method of using the pharmaceutical formulation.

Glaucoma is an eye disease that leads to irreversible visual disturbances. It is the fourth most common cause of blindness in the United States, the second most common cause of vision loss, and the most common cause of irreversible vision loss among African Americans. In general, the disease is characterized by progressive neuropathy due to deleterious effects resulting from at least partially increased intraocular pressure on the optic nerve. Open angle glaucoma contributes about 90% of all primary glaucoma and is characterized by abnormally high resistance to fluid (aqueous humor) drainage from the eye. Normal tolerance is necessary to maintain sufficient intraocular pressure to maintain eye shape for vision preservation. This resistance is provided by the trabecular strain, and the composite tissue consists of specific endothelial cells, connective tissue beams, and extracellular matrix. The resistance of the fibrous strain is usually such that the intraocular pressure, ie the pressure outside the eye, is ~ 16 mmHg at the same rate (2.5 μl / min) that the aqueous water is produced. Very high pressures (eg 70 mmHg) can cause blindness in just a few days. P. L. Kaufman and T. W. Mittag, "Medical Therapy Of Glaucoma," Ch. 9, Sec. II (pp. 9.7-9.30), in P. L. Kaufman and T. W. Mittag (eds.): Glaucoma (Vol. 7 of S. M. Podos and M. Yanoff (eds): Textbook of Ophthalmology Series). London, Mosby-Year Book Europe Ltd. (1994); A. C. Guyton, Textbook of Medical Physiology (W. B. Saunders Co., Sixth Ed.), Pp. See 386-89 (1981).

Recently, a large number of therapeutic agents have been used across a broad spectrum of chemical classes to approach the treatment of glaucoma and the regulation of elevated intraocular pressure.

Most often the type of drug used to reduce intraocular pressure is dependent on inhibition of intraocular water formation (eg beta-blockers, α2 adrenergic agonists, carbonic anhydrase inhibitors) or an increase in uveoscleral outflow (prostaglandin homologues). There is currently no conventional use of anti-glaucoma drugs that directly act on the trabecular line. Pilocarpine reduces fluid resistance secondaryly through the trabecular column, based on netting deformation as a result of drug-induced muscle contraction, but its use requires the need for three to four doses daily and local side effects, In particular limited by pupil narrowing. Epinephrine apparently acts directly on netting cells to increase ease through the β2 adrenergic receptor-mediated pathway, but only moderate efficacy in receptor-mediated local and systemic side effects, high frequency local allergy, and reactivity and in patients Due to its variability, it is rarely used clinically.

Fibrotomy is the most common form of glaucoma filtration surgery and exists as a first line treatment for surgical reduction of pharmacologically uncontrolled intraocular pressure in primary open angle glaucoma. This method reduces the intraocular pressure by forming a limbal fistula that drains the intraocular water into the subconjunctival space that forms the filter cloth. The success of this method is highly dependent on the pharmacological control of wound healing.

A major development in surgical treatment of glaucoma is the use of metabolic agents to prevent scarring after glaucoma filtration. Postoperative scarring of the bleb is the most important factor in determining the short and long term results of modern glaucoma filtration surgery. Metabolite mitomycin C (MMC) and 5-fluorouracil (5-FU) are the most widely used agents for suppressing the failure and scarring of filter cloth. In many past studies, conventionally performed trabeculectomy showed a failure rate of less than 30% within 3 months after surgery. To reduce the incidence of these detrimental complications, various methods have been studied to prevent spontaneous scarring of the blebs, most of which are anti-metabolic drugs-ie, 5-fluorouracil, the two most widely used cytotoxic agents. (5-FU) or mitomycin C (MMC) is treated with either preoperative or postoperative application.

Despite its apparent long-term effects on long-term filtration, the application of cytotoxic drugs to surgical eye increases the incidence of severe complications, such as the simultaneous increase in vision threat complications. MMC and 5-FU show a high incidence of severe post-application complications; Its side effects mainly affect the corneal epithelium, and its clinical use is limited by severe pain and anxiety in the patient. Not enough methods have been established for patients to achieve long term surgical outcomes after surgery with minimal or no side effects.

U.S. Patent Nos 6,586,425; 6,110,912; And 5,798,380 disclose a method of treating glaucoma using a compound that acts on the actin filament preservation of the eye to increase intraocular water outflow. The patent also specifically describes kinase inhibitors and latrunculin-A, latrunculin-B, swinholide-A and jasplatinol, which specifically regulate the interaction with membranes at or below the actin cytoskeleton in the trabecular line. The lead is disclosed. Perturbation and associated adhesion of the cytoskeleton reduces intraocular pressure by reducing the resistance of the fibrous strain to fluid flow.

Laterunculin is soluble in dimethyl sulfoxide (DMSO), but due to the very lipophilic nature of the large macrocyclic compound latrunculin, its water solubility is very low. DMSO is not acceptable for ophthalmic formulations for use in humans. Latrunculin-B is commonly dissolved in DMSO as a stock solution and stored at −20 ° C. for long term stability (Okka, et al., Trans. Am. Ophthalmol. Soc. 102: 251-259 (2004)). Known liquid ratunculin formulations are unacceptable in pharmaceutical formulations due to one or more of the following problems: unwanted side effects such as toxicity due to excipients or auxiliaries, low solubility of ratunculin without the use of DMSO, and Poor stability due to degradation of latrunculin over time.

There is a need for pharmaceutical formulations that can be used to treat glaucoma, to control wound healing after trabeculectomy, and to treat other diseases or disorders affected by the maintenance of actin cytoskeleton.

Summary of the Invention

The present invention relates to an aqueous pharmaceutical formulation comprising one or more ratunculins, which formulation does not contain a significant amount of dimethyl sulfoxide. The present invention provides an aqueous pharmaceutical formulation comprising one or more latrunculins and one or more agents that increase the solubility of latrunculin in an aqueous medium. The formulation does not contain a significant amount of any unacceptable medicament for pharmaceutical, especially ophthalmic use. The formulation provides ratunculin with sufficient activity for therapeutic use and is stable for at least 6 months at refrigeration temperature.

The present invention is directed to maintaining at least 200 to 400 mOsm / kG stiffness at one or more latrunculins in amounts of 0.001 to 2% w / v, nonionic surfactants in amounts of 0.01 to 2% w / v, and pH 4 to 8 It relates to an aqueous pharmaceutical formulation comprising an stiffener, wherein the latrunculin, surfactant and stiffener are miscible with the formulation and the formulation does not contain a significant amount of dimethyl sulfoxide.

In one embodiment, the aqueous pharmaceutical formulation comprises one or more latrunculins in an amount of 0.001-2% w / v, 1-100 mM buffer, 0.01-2% w / v, suitable for maintaining pH 4-6. Nonionic surfactants, and stiffeners for maintaining stiffness between 200 and 400 mOsm / kG. Preferred buffer is citrate buffer. Preferred stiffeners are mannitol and dextrose.

In another embodiment, the aqueous pharmaceutical formulation includes one or more latrunculins in an amount of 0.001 to 2% w / v, 5 to 10% ethanol, and an stiffener to maintain stiffness between 200 and 400 mOsm / kG. do. The formulations optionally include 1 to 100 mM buffer to maintain the pH between 4 and 8.

In another embodiment, the aqueous pharmaceutical formulation comprises one or more latrunculins in an amount of 0.001 to 2% w / v, 1 to 10% polypropylene glycol, 0.02 to 0.25% poloxamer, 0.1 to 1% polysorbate, And an stiffener for maintaining stiffness between 200 and 400 mOsm / kG, wherein the pH of the formulation is between 4 and 8.

In another embodiment, the aqueous pharmaceutical formulation comprises 0.001-2% latrunculin, cyclodextrin, 0.01-0.5% preservative, and an stiffener for maintaining stiffness at 200-400 mOsm / kG, wherein the formulation The pH of the water is 4-8.

The present invention provides a method for reducing intraocular pressure, a method for treating glaucoma, a method for inhibiting wound healing after trabeculectomy, and a method for inhibiting angiogenesis. The method comprises administering to a subject in need thereof an aqueous pharmaceutical formulation of the invention in an amount effective to alter actin cytoskeleton, for example by inhibiting actin polymerization.

Detailed description of the invention

Unless listed, the% amount herein refers to% (w / v).

The inventors have found an aqueous lathrunculin formulation that does not require the use of DMSO. We have found a non-DMSO aqueous lathrunculin formulation that is stable for a sufficient time at room temperature and refrigeration temperature. The inventors have found an aqueous lathrunculin formulation useful for the treatment of certain conditions such as glaucoma by inhibiting the polymerization of actin filaments. We have found a suitably tolerable aqueous rattunculin formulation for use in the eye.

The present invention relates to aqueous pharmaceutical formulations comprising macrocyclic compounds, in particular latrunculin and latrunculin homologues. The present invention provides formulations comprising one or more agents that increase the solubility of latrunculin in an aqueous medium. The formulations do not contain significant amounts of unacceptable agents for pharmaceutical, especially ophthalmic use. The present invention provides a stable aqueous formulation of ratunculin; The formulations are suitable for therapeutic use and are stable under normal use storage conditions for extended periods of time.

Aqueous pharmaceutical formulations of the invention, except for the use of inadequate dissolution of adjuvants such as methanol and dimethylsulfoxide, may cause toxicological consequences and tissue damage when used in humans or mammals for extended periods of time. Such pharmaceutical formulations contain latrunculin in aqueous solution at sufficient concentrations and exhibit pharmacological effects in reducing intraocular pressure in mammals. In topical administration, one to two drops of such formulation are delivered to the ocular surface one to four times per day, as determined by one of ordinary skill in the art. Such aqueous pharmaceutical formulations are non-irritant, acceptable to the eye, and suitable for many infusions.

Latrunculin is a cytoskeletal active macrolide. Latrunculin is a specific and potential actin-dividing agent that sequesters monomeric G-actin causing degradation of actin filaments. Natural latrunculins are marine sponges such as Latrunculia magnifica, Negombata magnifica, and Spongia mycofijiensis, and nudibranche, For example from Chromodoris lochi. Ratunculin homologues can be synthesized (AB Smith III et al., J. Am. Chem. Soc. 1992, 114, 2995-3007); JD White and M. Kawasaki, J Org.Chem. 1992, 57, 5292- 5300; A. Fuerstner et al., Angew. Chem. Int. Ed. 2003, 42, 5358-5380].

As used herein, latrunculin refers to the natural latrunculin and latrunculin homologues. Preferred latrunculin of the present invention is latrunculin B, latrunculin A, des-methyl latrunculin B, or a pharmaceutically acceptable salt, tautomer, solvate, or hydrate thereof.

Ratrunkullin B

Ratrunkullin A

Death-methyl latrunculin B

Ratrunculin homologues as used herein are described in [D. Blasberger et al., Liebigs Ann. Chem. 1171-1188 (1989); Fuersner et al., PNAS, 102: 8103-8108 (2005); And U.S. As described in patent application US2006-0217427, it refers to a synthetic compound whose structure is similar to that of natural ratunculin, the contents of which are incorporated herein by reference.

The present invention relates to an aqueous pharmaceutical formulation comprising one or more ratunculins, which formulation does not contain a significant amount of dimethyl sulfoxide. As used herein, “equivalent weight” refers to greater than 0.1%, preferably greater than 0.01%, more preferably greater than 0.001%. The aqueous pharmaceutical formulation of the present invention contains no more than 0.1%, preferably more than 0.01%, more preferably more than 0.001% v / v of DMSO. In a preferred embodiment, the aqueous pharmaceutical formulation of the present invention does not contain any dimethyl sulfoxide.

The present invention relates to an aqueous pharmaceutical formulation comprising at least one latrunculin in an amount of 0.001 to 2% w / v and an stiffener for maintaining stiffness between 200 and 400 mOsm / kG, wherein The pH is 4 to 9 and the formulation does not contain significant amounts of dimethyl sulfoxide. When the pharmaceutical formulation is used ophthalmically, the pH of the formulation is preferably 4 to 8, and the rigidity is preferably 220 to 380 mOsm / kG.

The present invention relates to one or more latrunculins in an amount of 0.001 to 2% w / v, 1 to 100 mM of buffer, 0.01 to 2% w / v, suitable for maintaining a pH of 4 to 8, preferably 4 to 6 An aqueous pharmaceutical formulation comprising a nonionic surfactant and a stiffener for maintaining stiffness between 200 and 400 mOsm / kG. Such pharmaceutical formulations do not contain significant amounts of DMSO and preferably contain up to 5% (v / v) ethanol, more preferably up to 2%, up to 1% ethanol.

It is important that the components of the formulation are compatible with each other. Compatibility as used herein refers to physical miscibility and chemical miscibility. Physical miscibility means that the components do not form precipitates or coacervates and do not cause phase separation, sedimentation or discoloration. Chemical miscibility means that the components do not cause degradation of latrunculin or inactivate its biological activity.

Surfactants (surfactants) suitable for the present invention may be nonionic or ionic. However, ionic surfactants are not preferred. Long-term use of cationic agents, especially cationic surfactants, is well known to cause corneal epithelial damage. Anionic surfactants are often immiscible with other components of the formulation.

Preferred surfactants for the present invention are nonionics. Suitable nonionic surfactants include, but are not limited to, polysorbate 80, polysorbate 60, polysorbate 20, tyloxapol, polyoxyl stearate, glyceryl monostearate, polyoxyl castor oil, polyethylene glycol car Prill triglycerides, and poloxamers. Preferred nonionic surfactants are polysorbates and poloxamers. Such surfactants are nonionic alkali oxide condensates of organic compounds containing hydroxyl groups. Nonionic surfactants increase the solubility of latrunculin. Nonionic surfactants often protect latrunculin molecules from chemical degradation by holding them in a micellar environment, which improves physical and chemical stability. The concentration of surfactant (s) in the formulation is about 0.01 to 2%, preferably 0.05 to 1.5%, more preferably 0.1 to 1% (w / v). Appropriate concentrations of the surfactant are determined by the solubility of latrunculin in the presence of the surfactant, by the neutralization of the bactericidal effect on the accompanying preservatives, and / or by the concentrations that can cause inflammation in humans. Pharmaceutical formulations prepared in the presence of a nonionic surfactant can be adjusted to a target pH of 4-8 without compromising the solubility of latrunculin.

Nonionic surfactants are not all suitable for ratunculin formulations because they are incompatible with ratunculin or other ingredients in the formulation or are not suitable for use in humans. For example, nonionic surfactants such as sorbitan monostearate, sorbitan trioleate, sorbitan monooleate, polyethylene glycol glyceryl cocoate, and oleate surfactants are miscible with ratuncurline formulations. This is because, when mixed with other ingredients in the formulation, these surfactants do not form a clear solution, but form coacervates and / or precipitates. Further, the nonionic surfactant TRITON ^® (polyoxyethylene octylphenyl ether) is not suitable for use in humans.

The concentration of latrunculin (s) in the aqueous formulation is usually from 0.001 to 2%, preferably from 0.005 to 0.2%, more preferably from 0.005 to 0.1%, more preferably from 0.005 to 0.02% (w / v). to be.

Buffers suitable for maintaining pH between 4 and 8 include phosphate, citrate buffer, acetate buffer, maleate buffer, tartarate buffer or combinations thereof. Preference is given to phosphate buffers or citrate buffers. For long term stability, the formulation preferably has a pH of 4-6. Buffers suitable for maintaining pH between 4 and 6 include citrate buffers, acetate buffers, citrate / phosphate buffers, maleate buffers, tartarate buffers or combinations thereof. Suitable concentrations of the buffer are 1 to 100 mM, preferably 5 to 50 mM, more preferably 5 to 25 mM, and most preferably 10 to 20 mM.

The stiffener is present in an amount to achieve stiffness between 200 and 400, preferably between 220 and 380, more preferably between 250 and 340 mOsm / kG. The stiffener can be nonionic or ionic. Nonionic stiffeners are preferred because they are often more miscible with surfactants than ionic stiffeners. Nonionic tonic agents include diols such as glycerol, mannitol, erythritol; And sugars such as dextrose. Other nonionic stiffeners such as glycerol, polyethylene glycol, propylene glycol, which also act as cosolvents, can also be used. The nonionic stiffener is usually 2 to 20%, preferably 3 to 10%, more preferably 3.5 to 5% (w / v). Preferred nonionic agents are mannitol and dextrose in amounts of 2 to 6%.

The tonicity agent may also be an ionic agent such as sodium chloride, potassium chloride, or a balanced salt solution. The ionic stiffener may be present at 0.5 to 0.9%, preferably 0.6 to 0.9% (w / v).

Surfactants, stiffeners, cosolvents, and any other ingredients introduced into the formulation must have good solubility in water, be miscible with the other ingredients, and have a mild effect on the final viscosity of the formulation. The formulations must have a suitable final viscosity that can be delivered as a topical drop using a typical ophthalmic dropper bottle and that is filtration-sterile. The formulation is preferably a clear solution free of any precipitate.

The pharmaceutical formulation of the present invention optionally comprises a chelating agent. Chelating agents are substances that can form several coordination bonds with metal ions. Chelating agents provide a wide range of binders that regulate metal ions in aqueous systems. By forming stable water soluble complexes with polyvalent metal ions, chelating agents interfere with the usual reactivity of metal ions to prevent unwanted interactions. Ethylenedinitrotetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and N, N-bis (carboxymethyl) glycine (NTA) are examples of chelating agents for the present invention. EDTA (ethylenediamine tetraacetate) is the preferred chelating agent.

In many countries, health regulations require that multi-administered ophthalmic formulations include preservatives. Many of the well known preservatives used in some other ophthalmic formulations cannot be used in the present invention, which is not considered safe for use in repeated eyes or that interacts with the surfactants used herein to sterilize the preservatives. This is because they form complexes that reduce activity. In one embodiment, benzalkonium chloride is used as a safe preservative; Preferably benzalkonium chloride is used with EDTA. Other suitable preservatives include benzyl alcohol, methyl parabens, propyl parabens, thimerosal, chlorobutanol, and benzethonium chloride. Typically, such preservatives are used at levels of 0.001 to 1%, preferably 0.01 to 0.25%, most preferably 0.05 to 0.2% (w / v).

In one embodiment, the pharmaceutical formulation comprises 0.5 to 0.9% ionic stiffness modifier, such as sodium chloride; The formulation comprises 1 to 100 mM of additional buffer (such as sodium phosphate and / or sodium citrate and citric acid), nonionic surfactant in the range of 0.01 to 2%, chelating agent in the range of 0.005 to 0.5% w / v, and pH And modulators. Such aqueous compositions have a stiffness of 250 to 350 mOsm / kG and are formulated at pH 4 to 6.

In one embodiment, the pharmaceutical formulation comprises 4-5% nonionic stiffeners such as mannitol; The formulation comprises a buffer in the range of 5 to 50 mM (such as sodium phosphate and / or sodium citrate and citric acid), a surfactant in the range of 0.01 to 2%, a chelating agent in the range of 0.005 to 0.5% w / v, and a pH adjusting agent. do. Such aqueous compositions have a stiffness of 250 to 350 mOsm / kG and are formulated at pH 4 to 6. The formulation optionally comprises a preservative in the range of 0.001 to 0.1% w / v.

In one embodiment, the pharmaceutical formulation comprises 0.001 to 2% w / v of latrunculin, 0.1 to 2% of polysorbate 80, and an stiffener to maintain stiffness between 200 and 400 mOsm / kG. . The formulation optionally comprises 1-100 mM of buffer for maintaining a pH of 4-6. Suitable buffers include phosphate, citrate buffer, acetate buffer, maleate buffer, tartarate buffer or combinations thereof. Preference or citrate buffer is preferred.

The invention also includes an aqueous solution comprising at least one latrunculin in an amount of 0.001 to 2% w / v, 5 to 25% ethanol (v / v), and a stiffener to maintain stiffness between 200 and 400 mOsm / kG. It relates to a pharmaceutical formulation. In ophthalmic applications, the ethanol concentration is preferably 5-10%. The formulation optionally comprises 1-100 mM of buffer for maintaining a pH of 4-8. Suitable buffers include phosphate, citrate buffer, acetate buffer, maleate buffer, tartarate buffer or combinations thereof. Preference is given to phosphate buffers or citrate buffers. The formulation does not contain significant amounts of DMSO. In one embodiment, the formulation comprises 0.005 to 0.02% latrunculin, 5 to 10% ethanol, and 0.5 to 0.9% sodium chloride.

The invention also relates to one or more latrunculins in an amount of 0.001 to 2% w / v, 1 to 10% (v / v) propylene glycol, 0.02 to 0.25% (w / v) poloxamer, and rigidity of 200 to 400 mOsm It relates to an aqueous pharmaceutical formulation comprising an stiffener for maintaining at / kG. The formulation optionally comprises 1 to 100 mM of buffer, and / or 0.1 to 1% of a surfactant such as polysorbate 80 to maintain a pH between 4 and 8. Suitable buffers include phosphate, citrate buffer, acetate buffer, maleate buffer, tartarate buffer, or combinations thereof. Preference is given to phosphate buffers or citrate buffers. The formulation does not contain any DMSO.

The invention also relates to at least one latrunculin in an amount of 0.001 to 2% w / v, cyclodextrin in an amount of 0.005 to 5%, preferably 0.01 to 2% w / v, benzyl chloride in an amount of 0.01 to 0.5% w / v An aqueous pharmaceutical formulation comprising a preservative such as alconium and an stiffener for maintaining stiffness between 200 and 400 mOsm / kG. Cyclodextrins that form complexes with latrunculin and increase the water solubility of latrunculin are suitable for the present invention. For example, 0.01-2% of hydroxypropyl-beta cyclodextrin or 0.01-1% of hydroxypropyl-beta cyclodextrin is included in the aqueous formulation. The formulation optionally comprises 1-100 mM of buffer for maintaining a pH of 4-8. Suitable buffers include phosphate, citrate buffer, acetate buffer, maleate buffer, tartarate buffer or combinations thereof. Preference is given to phosphate or citrate buffers. The formulation does not contain significant amounts of DMSO. The formulation may optionally be TWEEN ^® (polysorbate) -20, TWEEN ^® (polysorbate) -40, TWEEN ^® (polysorbate) -60, Span (sorbitan monooleate) -20, Span (sorbitan) Surfactants such as monooleate) -40, tyloxapol, polyvinyl pyrrolidone, and polyvinyl alcohol.

The present invention also relates to an emulsion comprising a microemulsion or a dilution emulsion, wherein the ratunculin is formulated with an adjuvant to form a uniform, reproducible and strong pharmaceutical formulation that is physically and chemically stable. Suitable auxiliaries include surfactants, emulsifiers, and pharmaceutically acceptable suitable vegetable or synthetic oils. The emulsion is formulated in an aqueous solution at a pH of 4-8, preferably 4-7, more preferably 4-6. The formulation optionally comprises 1-100 mM of buffer for maintaining a pH of 4-8. Suitable buffers include phosphate, citrate buffer, acetate buffer, maleate buffer, tartarate buffer or combinations thereof. Preference is given to phosphate or citrate buffers. The formulation does not contain significant amounts of DMSO. The emulsion optionally contains a suitable preservative and one or more antioxidants. Such emulsions can be prepared using conventional emulsification techniques including, but not limited to, high shear homogenization, ultrasound, high pressure homogenization, high pressure emulsification after mechanical stirring, or microfluidization or fluid-fluid interactions. have.

In one embodiment, the emulsion formulation comprises 0.001 to 2% latrunculin, 1 to 10% castor oil or 10 to 20% mineral oil, 1 to 5% carbomer or 1 to 10% cetyl alcohol, 0.1 to 1 % Polysorbate 80, 0.1-1% glyceryl monostearate or 0.1-2% polyoxyl stearate, and a tonicity agent such as glycerine or propylene glycol to maintain stiffness between 200 and 400 mOsm / kG; The pH of the formulation is 4-7.

The pharmaceutical formulations of the present invention are stable for at least 3 months, preferably at least 6 months, more preferably at least 12 months under cold storage temperatures. Stability as used herein refers to at least 70%, preferably at least 80%, more preferably at least 90%, ratunculin molecules that remain completely without degradation.

The pharmaceutical formulations of the present invention are suitable for storage at refrigeration temperature or room temperature, but are not stable when applied to the freeze freezing cycle because of the non-homogeneity of the ratunculin molecule and the change in the preservative effect of the formulation.

The pharmaceutical formulations of the present invention are prepared by sterile technique or finally sterilized. The purity of all materials used in the preparation exceeds 90%. The solution of the present invention is a ratunculin, buffers, stiffness modifiers, surfactants, chelating agents; Optionally, it is prepared by thoroughly mixing the nonionic polymer, complexing agent, solubilizer, preservative and antioxidant.

The pharmaceutical formulation may preferably be sterilized by filtering the formulation through a sterile grade filter of 0.22 micron nominal pore size. Pharmaceutical formulations may also be sterilized by final sterilization using one or more sterilization techniques, including but not limited to heat treatment or radioactive sterilization treatment, or using pulsed light to prepare a sterile formulation.

In one embodiment, the pharmaceutical formulations of the present invention are administered partially (eg topically, anteriorly, or via transplantation) to the eye in the form of an ophthalmic preparation. The pharmaceutical formulation may be mixed with an ophthalmically acceptable viscosity enhancer or penetration enhancer to form an ophthalmic suspension or solution. The pharmaceutical formulation can be used without further dilution or any other promotion.

Glaucoma is an eye disease that leads to irreversible vision damage. Open angle glaucoma is characterized by abnormally high resistance to fluid (relative water) discharge from the eye. Adhesion between cells of the fibrous line is a major determinant of resistance to flow. The pharmaceutical formulations of the present invention cause transient pharmacological fluctuations of cell adhesion mainly through regulation of interaction with the membrane or disruption of the associated cytoskeletal structure. This agitation of adhesion reduces the resistance of the fibrous strain to fluid flow, thereby reducing intraocular pressure in a therapeutically useful manner.

The pharmaceutical formulation of the present invention is useful for controlling wound healing after trabeculectomy. Pharmaceutical formulations are generally less toxic to corneal endothelial cells than metabolites such as 5-fluorouracil or mitomycin C. The pharmaceutical formulation inhibits actomyosin-induced contractility, leading to degeneration of the actin microfilament system and fluctuation of its membrane anchorage, which weakens cell-extracellular matrix adhesion. This property inhibits wound healing, reducing postoperative blister failure.

Angiogenesis is characterized by the development of angiogenesis from existing blood vessels and plays an important role in physiological processes such as embryogenesis, wound healing and female reproductive function, and pathophysiological conditions including cancer, rheumatoid arthritis and diabetic retinopathy. Do it. Tumor growth and metastasis are critically dependent on angiogenesis. Angiogenesis is a multistep process that involves mobilizing, proliferating and blocking stabilizing endothelial cell (EC) cytoskeleton. Applicants understand that the interaction between cytoskeleton and apoptosis is involved in the intracellular pathways through which neovascularization occurs. The pharmaceutical formulations of the present invention are useful for inhibiting angiogenesis and treating tumors.

Cell division inhibitory drugs interfere with antidiuretic responses, strongly indicating that cytoskeletal integrity is important for this function. This role of the cytoskeleton in regulating epithelial transport is a necessary step in the displacement of the water channel containing the particle mass and its delivery to the apex. The regulation of actin cytoskeleton is important for the regulation of fluid transport. Osmotic-dependent reorganization of the cytoskeleton and expression of certain pressure proteins are important components of the regulatory system involved in the adaptation of medulla cells to osmotic pressure. The pharmaceutical formulations of the present invention are useful for controlling epithelial function and regulating fluid transport.

The present invention provides a method for reducing intraocular pressure, a method for treating glaucoma, a method for inhibiting wound healing after trabeculectomy, a method for inhibiting angiogenesis, a method for treating cancer, and a method for controlling epithelial function and controlling fluid transport. The method comprises administering a pharmaceutical formulation of the invention to a subject in need thereof in an amount effective to alter actin cytoskeleton, such as by inhibiting actin polymerization.

The pharmaceutical formulations of the present invention are useful as intraocular pressure reducing agents and are therefore useful for the treatment or prevention of glaucoma or related ophthalmic conditions.

The pharmaceutical formulations of the present invention are useful for the treatment or prevention of neurodegenerative diseases as a result of increased intraocular pressure and damage to the optic nerve.

The pharmaceutical formulations of the present invention can also be used in the modes and routes of administration known to those skilled in the art, in the areas of cosmetics for wrinkle reduction, in areas of platelet embalming, and in areas of vascular spasm and smooth muscle spasms.

The pharmaceutical formulations disclosed herein may be administered topically to the eye of the patient in any suitable manner, but are preferably administered in the form of drops, sprays or gels. Alternatively, the pharmaceutical formulation may be applied to the eye via liposomes. In addition, the pharmaceutical formulation may be injected into the tear film via a pump-catheter system. In another embodiment, the pharmaceutical formulation is in a continuous or selective-release device, such as, but not limited to, Ocusert ™ system (Alza Corp., Palo Alto, Calif.) Or Retisert (Bausch & Lomb, Rochester, NY) It is contained in the same membrane as used. In a further embodiment, the pharmaceutical formulation may be contained in, delivered by, or attached to an eye contact lens. Another embodiment of the present invention includes a pharmaceutical formulation contained in a swab or sponge that can be applied to the eye surface. Another embodiment of the present invention includes a pharmaceutical formulation contained in a liquid spray that can be applied to the eye surface. Another embodiment of the invention includes infusion of the pharmaceutical formulation directly into the tear tissue or directly on the eye surface.

In one embodiment, the pharmaceutical formulation is administered systemically to the subject. The term systemic as used herein includes subcutaneous injection, intravenous, intravitreal injection, intracameral injection, subconjunctival injection, topical administration and oral administration.

Intravitreal delivery can include single or multiple intravitreal injections, or via implantable intravitreal devices that release cytoskeletal target compounds with sustained capacity. Intravitreal delivery may also include delivery applied directly to the vitreous as an adjunct of intraocular irrigation solution during surgical palpation or during a surgical procedure. Similar approaches may be made for subconjunctival or retrobulbar injection.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Without further elaboration, it is contemplated that one of ordinary skill in the art, using the previous teachings, can use the invention to the fullest extent. Accordingly, the specific preferred embodiments below are to be considered as illustrative only and are not limited in any way to the rest of the specification.

1 shows intraocular pressure (IOP,% baseline) versus time post-treatment of Dutch-belted rabbits treated with 0.02% latrunculin B in 5% ethanol formulation, and excipients.

2 shows intraocular pressure (IOP,%) of Dutch-belt rabbits treated with 0.02% latrunculin B in phosphate-buffered saline solution (pH ˜7) containing propylene glycol, poloxamer 407 and polysorbate 80 (pH ˜7). Baseline) versus time after treatment.

FIG. 3 shows citrate-buffer (pH ˜5.5) containing 4.5% w / v mannitol, 1% w / v polysorbate 80, 0.05% w / v disodium edate and 0.01% w / v benzalkonium chloride. ) Intraocular pressure (IOP,% baseline) vs. time post-treatment of Dutch-belt rabbits treated with 0.02% latrunculin B, and excipients.

4 shows intraocular pressure (IOP,% baseline) versus time post-treatment of Dutch-belt rabbits treated with 0.02% des-methyl latrunculin B in 5% ethanol formulation, and excipients.

FIG. 5 shows citrate-buffer (pH ˜) containing 4.5% w / v mannitol, 1% w / v polysorbate 80, 0.05% w / v disodium edate and 0.01% w / v benzalkonium chloride. 5.5) intraocular pressure (IOP,% baseline) vs. time post-treatment of Dutch-belt rabbits treated with 0.1% cis-des-methyl latrunculin B, and excipients.

Example  1.5% ethanol excipient Formulation  medium Latrunkullin  B, 0.02% Formulation  Produce

After adding 250 μl of 200 proof ethanol to a vial containing 1 mg of ratunculin B, the solution was mixed at ambient temperature. 750 μl of a phosphate-buffered saline solution with a pH of about 7 was added to this solution; The solution was mixed at ambient temperature for about 5 minutes. The resulting solution was clear and 0.1% relative to latrunculin B. The solution was used on its own or diluted to a concentration of 0.02% using a phosphate-buffered saline formulation.

Example  2. Aqueous Excipients Formulation  medium Latrunkullin  Preparation of B Solution

100 μl of propylene glycol was added to a vial containing 1 mg of latrunculin B, then the solution was mixed at ambient temperature. Ratrunculin B immediately became a solution. The solution was cooled in an ice bath to about 5 ° C. To this solution was added 450 μl of phosphate-buffered saline solution (pH ˜7) containing 0.25% w / v poloxamer 407 and mixed for 10 minutes while maintaining this temperature at about 5 ° C. To this solution was added 450 μl of phosphate-buffered saline solution (pH ˜7) containing 1% w / v polysorbate 80 and mixed at ambient temperature for 10 minutes. The resulting solution was clear and 0.1% relative to la trunculin B. The solution was used on its own or diluted to a concentration of 0.02% using phosphate-buffered saline solution (pH ˜7).

Example  3. Aqueous Excipients Formulation  medium Latrunkullin  B Formulation  Produce

50 μl of propylene glycol and 40 μl of glycerin were added to a vial containing 1 mg of ratunculin B, and then the solution was mixed at ambient temperature. Ratrunculin B became a solution. The solution was cooled in an ice bath to about 5 ° C. To this solution was added 910 μl of phosphate-buffered saline solution (pH ˜7) containing 0.2% w / v poloxamer 407 and 1% w / v polysorbate 80. After addition, the solution was mixed at 5 ° C. for about 10 minutes and then at room temperature for an additional 10 minutes. The resulting solution was clear and 0.1% relative to latrunculin B. The solution can be diluted by itself or to a concentration of 0.02% or less using phosphate-buffered saline solution (pH ˜7) containing 0.2% w / v poloxamer 407 and 1% w / v polysorbate 80 Used.

Example  4. Aqueous Excipients Formulation  medium Latrunkullin B of  Preparation of Preservatives

A vial containing 1 mg of ratunculin B contains 4.5% w / v mannitol, 1% w / v polysorbate 80, 0.05% w / v disodium edate and 0.01% w / v benzalkonium chloride One mL of about 10 mM citrate buffer (pH ˜5.5) was added and mixed for about 15 minutes at room temperature. The solution was clear and colorless. The solution was 0.1% relative to Ratrunculin B and had a pH of about 5.5. The solution is itself or citrate buffer containing 4.5% w / v mannitol, 1% w / v polysorbate 80, 0.05% w / v disodium edate and 0.01% w / v benzalkonium chloride (pH ˜5.5) was used to dilute to a concentration of 0.02% w / v or less (eg 0.005% w / v).

Solutions containing 0.02% (solution A) and 0.005% (solution B) latrunculin B were tested for stability. The degradation of latrunculin B was measured by HPLC. The results show that after 6 months storage at 2-8 ° C., Solution A retains 95% of the initial amount of latrunculin B and Solution B maintains 100%.

Example  5. 5% Ethanol Excipient Formulation  medium death - methyl Latrunkullin  B, preparation of 0.02% solution

To a vial containing 1 mg of des-methyl latrunculin B was added 250 μl of 200 proof ethanol and then the solution was mixed at ambient temperature. 750 μl of a phosphate-buffered saline solution with a pH of about 7 was added to this solution; The solution was mixed at ambient temperature for about 5 minutes. The resulting solution was clear and 0.1% relative to latrunculin B. The solution was used on its own or diluted to a concentration of 0.02% using a phosphate-buffered saline formulation.

Example  6. Aqueous Excipients Formulation  medium Sheath - death - methyl Latrunkullin  Preparation of B Solution

In a vial containing 1 mg of cis-des-methyl latrunculin B, 4.5% w / v mannitol, 1% w / v polysorbate 80, 0.05% w / v disodium edate and 0.01% w / v chloride 1 mL of about 10 mM citrate buffer (pH ˜5.5) containing benzalkonium was added and mixed for about 15 minutes at room temperature. The solution was clear and colorless. The solution was 0.1% relative to Ratrunculin B and had a pH of about 5.5. The solution is itself or citrate buffer containing 4.5% w / v mannitol, 1% w / v polysorbate 80, 0.05% w / v disodium edate and 0.01% w / v benzalkonium chloride (pH ˜5.5) was used to dilute to a concentration of 0.02% w / v or less.

Example  7. In Vivo Cytoskeletal Target Compounds Formulation  Effect on Intraocular Pressure

Animal Preparation and Apparatus: All in vivo experiments and evaluations of tolerability of formulations and studies of the effect on intraocular pressure of compounds in the formulations were conducted in Dutch-belt rabbits. Animals used for this assessment were individually housed in barns exposed to a normal, irreversible 12 hour light cycle and had free access to food and water. Intraocular pressure was measured using a commercially available TonoPen XL tonometer.

Experimental Protocol: Rabbits were removed from their cages and locked in cloth bags for several minutes prior to IOP measurements to minimize stress-induced increases in IOPs. The cornea of the rabbit was anesthetized with 0.25% proparacaine HCl. After waiting for about 2 minutes to allow the anesthetic to take effect, the eyelids were kept open with non-dominant hand, and Tono-Pen was touched several times in the center of the cornea and recorded. A pharmaceutical formulation containing latrunculin B or a control excipient was administered to each animal as one or more topical eye drop (s). Typically, 20 μl (2 drops × 10 μl each) were administered to the center of the cornea at about 30 second intervals using a pipette. Flickering to prevent maximization of penetration; An equal volume of the same test compound solution or excipient was instilled with both eyes. IOP measurements were taken before dropping injection (-1 hour, 0 hours) and after dropping injection for 1, 2-2.5, 3, 4.5 and 6 hours. After each series of measurements the animals were returned to cages. In addition to IOP measurements, a brief eye examination was performed before and during the course of administration and at the end of the study. The focus was on observing eye conditions, particularly ocular symptoms such as hyperemia, conjunctival edema, conjunctival discharge and corneal clouding.

result

1 shows the intraocular pressure reduction effect of Dutch-belt Levit treated with 0.02% latrunculin B in 5% ethanol formulation, and excipients.

FIG. 2 shows the intraocular pressure reduction effect of Dutch-belt rabbits treated with 0.02% latrunculin B in phosphate-buffered saline solution (pH ˜7) containing propylene glycol, poloxamer 407 and polysorbate 80, and excipients. (Example 3).

FIG. 3 shows citrate-buffer (pH ˜5.5) containing 4.5% w / v mannitol, 1% w / v polysorbate 80, 0.05% w / v disodium edate and 0.01% w / v benzalkonium chloride. ), The intraocular pressure-reducing effect of Dutch-belt rabbits treated with 0.02% latrunculin B, and excipients (Example 4).

FIG. 4 shows the intraocular pressure reduction effect of Dutch-belt rabbits treated with 0.02% des-methyl latrunculin B in 5% ethanol formulation, and excipients (Example 5).

FIG. 5 shows citrate-buffer (pH ˜5.5) containing 4.5% w / v mannitol, 1% w / v polysorbate 80, 0.05% w / v disodium edate and 0.01% w / v benzalkonium chloride. Intraocular pressure reduction effect of Dutch-belt rabbits treated with 0.1% cis-des-methyl latrunculin B, and excipients).

The present invention, and the manner and methods of making and using the same, are now described in these complete, clear, concise and accurate terms so that any person skilled in the art can make and use the same. The foregoing describes preferred embodiments of the invention and it is understood that changes may be made without departing from the scope of the invention as described in the claims. In order to particularly point out and explicitly claim the subject matter regarded as the present invention, the following claims complete the specification.

Claims (18)

At least one latrunculin in an amount of 0.001 to 2% w / v, a nonionic surfactant in an amount of 0.01 to 2% w / v, and an stiffener for maintaining stiffness at a pH of 4 to 8 at 200 to 400 mOsm / kG An aqueous pharmaceutical formulation comprising an aqueous pharmaceutical formulation wherein latrunculin, a surfactant, and a tonic agent are miscible with the formulation and the formulation does not contain significant amounts of dimethyl sulfoxide. The aqueous pharmaceutical formulation of claim 1, wherein the formulation does not contain greater than 0.001% v / v dimethyl sulfoxide. The aqueous pharmaceutical formulation of claim 1, wherein the formulation does not contain any dimethyl sulfoxide. The method of claim 1, wherein the nonionic surfactant consists of polysorbate, tyloxapol, polyoxyl castor oil, poloxamer, polyethylene glycol carfil triglycerides, polyoxyl stearate, glyceryl monostearate, and combinations thereof. An aqueous pharmaceutical formulation selected from the group. The aqueous pharmaceutical formulation of claim 1, wherein the nonionic surfactant is polysorbate, poloxamer, or a combination thereof. The aqueous pharmaceutical formulation of claim 1, further comprising 1 to 100 mM buffer suitable for maintaining a pH of 4 to 6. 7. The aqueous pharmaceutical formulation of claim 6 wherein the buffer is citrate buffer, acetate buffer, citrate / phosphate buffer, maleate buffer, tartarate buffer, or a combination thereof. 8. An aqueous pharmaceutical formulation according to any one of the preceding claims further comprising a chelating agent and / or preservative. The aqueous pharmaceutical formulation of claim 1 wherein the stiffener is a nonionic stiffener. 10. The aqueous pharmaceutical formulation of claim 9, wherein said nonionic stiffener is mannitol or dextrose. The aqueous pharmaceutical formulation according to any one of claims 1 to 10, wherein said formulation is stable for at least 6 months at 2-8 ° C. The aqueous pharmaceutical formulation of claim 1, wherein said surfactant is poloxamer in an amount of 0.02 to 0.25% w / v and said formulation further comprises 1 to 10% v / v propylene glycol. An aqueous pharmaceutical formulation comprising at least one latrunculin in an amount of 0.001 to 2% w / v, 5 to 10% v / v ethanol, and an stiffener for maintaining stiffness between 200 and 400 mOsm / kG, wherein An aqueous pharmaceutical formulation having a pH of the formulation of 4 to 8 and containing no significant amount of DMSO. An aqueous pharmaceutical formulation comprising one or more latrunculins, cyclodextrins, preservatives, and an stiffener for maintaining stiffness between 200 and 400 mOsm / kG in an amount of 0.001 to 2% w / v, wherein the pH of the formulation is An aqueous pharmaceutical formulation of 4 to 8 and containing no significant amount of DMSO. The aqueous pharmaceutical formulation of any one of claims 1 to 14, wherein the latrunculin is latrunculin A, latrunculin B, des-methyl latrunculin B, or a latrunculin homologue. The aqueous pharmaceutical formulation of claim 15 wherein said latrunculin is latrunculin A, latrunculin B, or des-methyl latrunculin B. The aqueous pharmaceutical formulation of claim 15 wherein said latrunculin is in an amount of 0.005 to 0.02% (w / v). A method of reducing intraocular pressure in a mammal comprising administering to the mammal in need thereof an aqueous pharmaceutical formulation according to any one of claims 1 to 17 in an amount effective to alter the actin cytoskeleton.

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